We collected peer-reviewed publications that investigated the impacts of MYB overexpression on the regulatory mechanisms of anthocyanidin biosynthesis using the Web of Science and PubMed databases up to 31 December 2020. An extensive literature search was performed for articles dated through 31 December 2020 using our systematic search terms: (MYB or MYB transcription factor*) and (overexpression* or ectopic* expression* or transgenic or heterologous) and (anthocyanin* or leucoanthocyanidin*). A total of 496 unique articles were sorted. Upon examination, 290 publications were excluded because they did not conform to the following criteria: the study did not relate to MYB transcription factors (35 articles); MYB genes were not overexpressed (112 articles); articles were reviews (13 articles); either treatment or control means were missing (6 articles); or articles did not report the outcome variables of interest (124 articles). Finally, 206 articles spanning 19 years (2001-2020) on MYB overexpression in the regulation of the anthocyanin biosynthetic pathway were contained in our meta-analysis. The details are described in Supplementary Table 1 and Supplementary Figure 1.

Multiple treatments from one article were deemed independent studies and represent a unit in the meta-analysis. Means and sample sizes were extracted for both MYB overexpression plants and corresponding non-transformed parentals from each of the individual studies. Biological replicates were referred to “sample sizes”. Where articles did not report those sizes, we determined the values conservatively as n = 1 if average values were not reported and n = 2 if average values were provided. When the sample capacity was given as a scale, the meta-analysis selected the minimum value. When the article included data for a time course, we selected the final time point as separate observation. We used GetData Graph Digitizer¹ to extract the data in the chart.

A meta-analysis was performed on the response of various variables in control and MYB transgenic plants to the modulation of anthocyanin accumulation. Each study was included in a meta-analysis, and the combined response of the transgenic plants was defined corresponding to non-transgenic plants to compare a treatment effect size, which was calculated as the natural logarithm of the response ratio (ln R) of the transgenic to non-transgenic (control plant means) (Hedges et al., 1999). Following formula was applied:

Here, Y_TC and Y_NC refer to the average values of responses from transformed (TC) plants and corresponding non-transformed (NC) parentals, respectively (Ma et al., 2017). The ln R-values are largely applied in meta-analyses of plant behaviors to obtain a standardized, unitless expression of the effects of variables and treatments, revealing biological significance (Borenstein et al., 2009; Ma et al., 2017). Across response ratios, the positive and negative treatment effects are properly balanced by log transformation, which maintains symmetry in the meta-analysis. The ln R-values greater than 0 imply an increase in the measured parameter after MYB overexpression and the ln R-values less than 0 imply that the measured parameter is decreased or unchanged. For ease of interpretation, the mean ln R was transformed into the percentage change using the following formula:

Overall, we calculated summary effect size for each of the 26 parameters as the weighted average of effect sizes from the initial studies. Apart from the effect size data, thirteen experimental variables or moderator variables was collected from each study. The response to anthocyanidin biosynthesis was modified by these variables. We used moderator variables to test for heterogeneity within the effect sizes. These moderators were of four categories: (I) anthocyanin and color: anthocyanin content change, color, color change, numbers of color types; (II) experimental materials: sample type, taxonomic class (dicot or monocot, or other) and genus of gene donor and recipient, whether the gene donor and recipient were the same genus; (III) character of foreign genes: overexpression type, number of genes transferred; and (IV) experimental conditions: whether experimental conditions were normal culture.

Each moderator variable included more than two levels (categories), and a level of a moderator was required to be documented in no less than three studies, with ≥ 2 published report. Categorical levels of moderators not meeting the stated criteria were classified as “other”. However, the level of “other” was also needed to contain data obtained from no less than three studies, with ≥ 2 published report.

We used Comprehensive Meta-Analysis (CMA) software (Version 3.0, Biostat, Englewood, NJ) for meta-analyses. Single studies were weighted using non-parametric variance methods:

Here, nTC and nNC refer to the number of samples of the TC and NC samples, respectively, and V ln R refer to the variance of the natural log of the response ratio R (Borenstein et al., 2009). The summary effect was assumed significant if its p-value was less than 0.05 and the 95% confidence intervals (CIs) did not overlap 0. We used the Q statistic to assess whether the effect value indicated heterogeneity, and to quantify the heterogeneity of the observed effect size. The true heterogeneity of the effect value did not exist when its I²-value was 0%, while the I²-value > 50% reflected unacceptable heterogeneity. A common variance among studies across moderator subgroups was supposed when its 95% CI did not overlap 0 and the p value was less than 0.05.

Because statistically significant studies are easy to publish than non-significant studies, and meta-analyses often focus on the published literature, and there can be publication bias in meta-analysis selection bias (Borenstein et al., 2009). Although published articles were accessed from multiple sources to avoid publication bias, it is unclear whether such bias exists. Therefore, we adopted four statistical methods in our study to test potential publication bias, which was quantified using the classic fail-safe N test Begg and Mazumdar (1983), Orwin (1983) rank correlation test, Egger’s regression intercept test (Egger et al., 1997) and Duval and Tweedie’s trim and fill calculation (Duval and Tweedie, 2000).

Within the classic fail-safe N test, seven of the 26 summary effects had a p-value ≥ 0.01, indicating that there was concern of bias (Table 1). The Begg and Mazumdar rank correlation test showed that only one summary effect had a p < 0.05, indicating the possibility of bias. Egger’s regression intercept test demonstrated that three summary effects may be biased. Egger’s regression intercept test demonstrated that there may be publication bias in three summary effects. Duval and Tweedie’s trim and fill analysis indicated that 22 of these effects may be affected by adjusted value. The adjusted value of flavonoid content, PAL, and ANR were closer to 0 than the primeval point estimates, while 19 of the 22 summary effects were farther from 0. Therefore, the three summary effects may have a bias effect. Using four quantitative methods, a total of 11 summary effects were evaluated, which may have publication bias. Two summary effects were detected by two methods, and none was detected by three or more methods. This shows that there was no significant bias in 26 summary effects, which was not an important influence on the results of this study.

As shown in Table 2, we found substantial heterogeneity (P-value < 0.100) and I²-value > 50% in 18 out of 26 summary effects values. Therefore, subgroup analysis can be used to search the possible sources of true heterogeneity among studies. In meta-analyses, effect size heterogeneity usually relates to the change in “true” effect sizes. However, heterogeneity and random error make up the “true” observed changes, and a P-value > 0.100 or I²-value < 25% does not essentially mean that real heterogeneity among studies does not exist. Insufficient sample size and large differences in test conditions may also result in a P-value > 0.100. Therefore, even if there was no heterogeneity in the statistical significance, we considered selecting a random effect model to perform the subgroup analysis of the summary effect sizes for different variables.

Figure 1 summarizes the average ratio of TC and NC plants of 26 indicators. Seventy-eight species documented in 206 publications were used in the meta-analysis. A total of 59 genera were used as donors, with Arabidopsis being the most studied for MYB genes, (91 studies), followed by Malus (56 studies) and Solanum (51 studies). Regarding the type of donors, the most examinations were obtained from dicots (554 studies). The diversity of receptor genera was less and it was observed in only 33 genera; Nicotiana was the most studied (225 studies), followed by Arabidopsis (123 studies) and Malus (52 studies). The highest number of studies (503 studies) was related to stably overexpressing MYB transcription factors and 105 studies discussed transient overexpression. Natural logs (ln R) of summary effect sizes are shown in forest plots (Figure 1), where the summary effect size and its CIs are represented in log scale, along with percentage changes calculated from reverse-transformed data.

Overall, MYB transformation had a statistically significant impact on 21 out of 26 measured plant parameters (Figure 1: p < 0.05, CIs did not cross-zero). The anthocyanin content, cyanidin content and delphinidin content were significantly affected by MYB overexpression. Cyanidin content was the most impacted (768%; Table 2). The contents of flavonol, proanthocyanidin, quercetin and kaempferol were increased by more than 53%, while flavonoid content was not affected by overexpressing MYB. Transformation did not affect pH or catalase (CAT) activity. In transgenic plants, malondialdehyde (MDA) content was reduced by 37%, whereas the activity of 1,1-diphenyl-2-picryl-hydrazyl (DDPH) was improved by 64% (Table 2). The expression of PAL was significantly increased in TC plants by 47%, while the expression of C4H and 4CL did not significantly respond to transformation. The expression of CHS, CHI and F3H was also significantly increased by 165%. MYB overexpression had statistically significant impacts on the regulation of late structural genes of anthocyanin biosynthesis, including FLS, F3′H, F3′5′H, DFR, ANS and UFGT. The expression of F3′5′H was the most impacted (882%), followed by ANS (412%) and DFR (340%). The expression of LAR and ANR were also improved by MYB transformation (Table 2).

By performing subgroup analysis of the 26 summary effects, we determined the categories or levels variables with significant effects on true heterogeneity. Moderating variables such as experimental conditions, experimental materials and characteristics of foreign genes were registered and used for subgroup analysis. Figures 2–8 and Supplementary Figures 2–8 show the effect of the moderator levels (categories) on the summary effects. We found substantial heterogeneity (P-value < 0.100) and I²-value > 50% in 18 out of 26 summary effect values (Table 2). In meta-analyses, the heterogeneity of the effect values usually refers to the change in “true” effect sizes and the change in “true” effect sizes usually represents the heterogeneity of the effect sizes.

However, heterogeneity and random error make up the “true” observed changes, and a P-value > 0.100 or I²-value < 25% does not essentially mean the absence of real heterogeneity among studies (Borenstein et al., 2009). Insufficient sample size and large differences in test conditions may also result in a P-value > 0.100. Therefore, even if there was no heterogeneity in the statistical significance, we considered selecting a random effect model to conduct the subgroup analysis of the summary effect sizes for different variables.

The overexpression of MYB in transgenic plants strongly promoted the contents of anthocyanin, cyanidin, delphinidin, flavonol, proanthocyanidin, quercetin and kaempferol (Figures 2, 3 and Supplementary Figures 2, 3). Compared with the decreased or unchanged anthocyanin content, the transgenic plants showed significant increase in the contents of anthocyanin, cyanidin and proanthocyanidin to 741, 18,468, and 387%, respectively (Figure 2 and Supplementary Figure 2A). When the anthocyanin content was unchanged, the transgenes had the greatest impact on the flavonol content (Figure 3A). However, the change in anthocyanin content did not show significant penalties in the contents of quercetin and kaempferol (Supplementary Figure 3). When the color of transgenic plant tissue was red or purple, the effects of MYB transformation on the contents of anthocyanin, cyanidin, and proanthocyanidin were more significant than those of other colors (Figures 2, 3B and Supplementary Figure 2A). In the red tissues, the contents of delphinidin and kaempferol were significantly improved by 115 and 111%, respectively (Supplementary Figures 1B, 3B). In the purple tissues, the MYB transformation had positive effects on the contents of flavonol and quercetin (Figure 3A and Supplementary Figure 3A). The cyanidin content was the most impacted, reaching 32.4×, followed by the anthocyanin content (8.4×) and delphinidin content (5.6×) (Figures 2, 3 and Supplementary Figures 2, 3). However, during a lighter color change, MYB transformation negatively impacted the anthocyanin content and cyanidin content, decreasing up to 69% and 98%, respectively (Figure 2 and Supplementary Figure 2A). However, when the color was unchanged, NC plants improved delphinidin content and flavonol content by 29.9× and 2.2×, respectively (Figure 3A and Supplementary Figure 2B).

With an increasing number of tissue colors in transgenic plants, the accumulation of anthocyanin content and cyanidin content increased significantly (Figure 2 and Supplementary Figure 2A). If the tissue number was 4, MYB transformation had the maximum effect on cyanidin and proanthocyanidin contents, which was more than sixteen times and thirty-two times that of the other number groups (Figure 3B and Supplementary Figure 2A). Significantly increased anthocyanin, cyanidin, and delphinidin contents were observed in the leaf tissues of transgenic plants as compared to other tissues (Figure 2 and Supplementary Figure 2). However, increased contents of flavonol, proanthocyanidin, quercetin and kaempferol contents were observed in calluses, roots, seedlings and flowers, respectively (Figure 3 and Supplementary Figure 3). Anthocyanin content and flavonol content were affected by the taxonomy of donor plants MYB genes from dicots showed significantly higher responses than those from other donor plants (Figures 2, 3A). Contrarily, Monocot donors showed 1.7 times higher contents of proanthocyanidin as compared to dicots (Figures 2, 3B). For receivers, dicots accumulated more anthocyanin content than monocot plants (Figure 2). Regarding the genera of donors, TC plants showed the maximum improvement in the contents of cyanidin, flavonol, proanthocyanidin and kaempferol for overexpressing MYB genes from Arabidopsis, whereas the most anthocyanin and delphinidin content responses were observed for Solanum (Figures 2, 3 and Supplementary Figures 2A, 3B). Nicotiana seems to be an appropriate MYB recipient genus, imparting at least 562% increase in the contents of anthocyanin, cyanidin, delphinidin and proanthocyanidin (Figures 2, 3A and Supplementary Figure 2). However, the flavonol, quercetin and kaempferol contents of recipient Arabidopsis were higher than those of other genera (Figure 3A and Supplementary Figure 3). When MYB genes were expressed homologously, TC plants showed a better effect on the content of cyanidin, flavonol and quercetin than heterologous expression (Figure 3A and Supplementary Figures 2A, 3A). In contrast, heterologous expression had a significant influence on the responses of the contents of anthocyanin, delphinidin, proanthocyanidin and kaempferol (Figures 2, 3B and Supplementary Figures 2B, 3B). Stable expression of MYB genes in plants better affected the contents of anthocyanin, cyanidin, flavonol, proanthocyanidin and quercetin than transient overexpression (Figures 2, 3 and Supplementary Figures 2A, 3A). Transfer of transient cells demonstrated a remarkable TC-induced improvement in the content of delphinidin and kaempferol, which was more than 21-fold and 3-fold than that of stable overexpression (Supplementary Figures 2B, 3B). Transfer of single genes had a greater impact on the content of delphinidin, proanthocyanidin, quercetin and kaempferol than multiple gene transfers (Figure 3B and Supplementary Figures 2B, 3). When two genes were transferred, the accumulation of cyanidin content and flavonol content were markedly increased, wherein the cyanidin content was eight times higher than that of single gene transformation (Figure 3A and Supplementary Figure 2A). More than three genes caused more anthocyanin content, which was more than 5.3 times that of single or two gene transfers (Figure 2). For environmental conditions, compared with normal culture, the positive effects of overexpressing MYB on flavonol and proanthocyanidin content were increased when grown in abnormal environments, whereas higher responses of anthocyanin and cyanidin contents were shown in normal environments (Figures 2, 3 and Supplementary Figure 2A).

No significant impact of DDPH activity on the change in anthocyanin content was demonstrated in TC plants (Figure 4B). For color, a purple color had a significant influence on DDPH activity after MYB transformation than that of red, while MDA content was not significantly altered by the color change (Figure 4). Regarding the number of colored tissues, one tissue provided the highest DPPH activity, which was improved by 272% (Figure 4B). The outcomes of transformation on DDPH activity were most evident in relation to the sampled tissues, with the TC-induced impact averaging 358 and 132% for roots and other tissues, respectively (Figure 4B). As donor plants, other donors showed more DDPH activity after transformation than dicots, with other genera showing the lowest MDA content when acting as donors (Figure 4). Similar to donor plants, DDPH activity was also induced when receiver plants were other receivers (Figure 4B). For the receptor genus, other genera showed a significant increase in DDPH activity relative to Arabidopsis, Nicotiana, Petunia, and Solanum (Figure 4B). Homologous expression resulted in higher DDPH as compared to heterologous systems (Figure 4B). When plants were grown in normal culture, the effect of MYB transformation on increasing the activity of DDPH was greater, whereas higher inhibition of MDA content was observed in an abnormal environment (Figure 4).

Overexpressing MYBs significantly upregulated the expression of PAL and C4H, resulting in the increased anthocyanin content of transgenic plants by at least 2.1-fold as compared to NC plants (Figure 5 and Supplementary Figure 4A). When the anthocyanin content was decreased, 4CL was downregulated by 63% in MYB transformation plants (Supplementary Figure 4B). The highest impact of MYB transformation on the downregulation of C4H was observed in red tissues of transgenic plants (Supplementary Figure 4A). Regarding the color change, PAL and 4CL were significantly upregulated by a deeper color of TC plants. However, the transcripts of C4H and 4CL decreased gradually due to reduction in color intensity by overexpression of MYB (Figure 5 and Supplementary Figure 4). The PAL gene was notably expressed in three tissue colors, whereas C4H and 4CL were not significantly changed by the number of color types (Figure 5 and Supplementary Figure 4).

The effects of transformation on PAL and C4H were the most significant in roots (Figure 5 and Supplementary Figure 4A). In other tissues, PAL, C4H, and 4CL were upregulated at least 2.4-fold relative to NC plants (Figure 5 and Supplementary Figure 4). C4H expression was rapidly decreased in petals (Supplementary Figure 4A). For donors, dicot plants were much more responsive to the expression of PAL than monocotyledonous plants, but donor plants did not significantly differ in C4H and 4CL expression (Figure 5 and Supplementary Figure 4). Other genera showed the highest increase in the transcription of C4H in donor plants (Supplementary Figure 4A). Although the expression of PAL and C4H was significantly induced in transformed Arabidopsis and other genera, C4H was inhibited when genes came from Petunia, or when donor and recipient plants were of the same genera, and MYB transformation significantly promoted the expression of C4H and 4CL (Figure 5 and Supplementary Figure 4). However, overexpressing MYB in heterologous systems positively regulated PAL expression (Figure 5). There was no notable difference between transient transformation and stable transformation in the expression levels of PAL, C4H and 4CL (Figure 5 and Supplementary Figure 4). For PAL expression, single gene transformation caused a considerable impact on TC plants, and normal culture was a more influential environmental condition than stress (Figure 5).

The effect of transformation on improving the expression levels of early-stage genes of anthocyanin biosynthesis was consistent across anthocyanin content changes. The expression of CHS, CHI, and F3H was the highest, causing the increased anthocyanin content of TC plants (Figure 6 and Supplementary Figure 5). For the color change of transgenic plant organs, we also noted a similar result from the early-stage genes, with the greatest effect in purple color, followed by red or purple and red (Figure 6 and Supplementary Figure 5). Similarly, in the anthocyanin content change, the dark color of overexpression plants significantly enhanced the expression of CHS, CHI and F3H, by 5. 5-, 3. 0-, and 4.2-fold, respectively (Figure 6 and Supplementary Figure 5). The expression levels of CHS, CHI and F3H were found to increase with increasing number of organ colors in TC plants, with the highest expression of early-stage genes in four tissue colors (Figure 6 and Supplementary Figure 5). In roots of TC plants, the expression of CHI and F3H was markedly upregulated by 11.9- and 10.2-fold, respectively, while CHS expression was the highest in other tissues (Figure 6 and Supplementary Figure 5). The effect of transformation on the influence of CHS and CHI expression was consistent across donors and receiver type, with their transcription being higher in dicots (Figure 6). In contrast, F3H expression was markedly induced when donors and receivers were monocot plants (Supplementary Figure 5). With MYB genes from Vitis, the donor genus had similar positive impacts on the expression of CHI and F3H, upregulated by 13.5- and 80.0-fold, respectively, as compared to genes from solanum (Figure 6B and Supplementary Figure 5). However, Vitis seems to be a useful MYB donor, causing at least 6.7-fold increase in CHS expression (Figure 6A). Although there was no contrasting effect on the expression of CHS, CHI and F3H under recipient Petunia and Solanum, some differences can be distinguished in Arabidopsis, Nicotiana and other genera (Figure 6 and Supplementary Figure 5). Although the expression systems of plants considerably enhanced the expression of CHS, CHI and F3H, heterologous or homologous expression demonstrated little difference among genes (Figure 6 and Supplementary Figure 5). Regarding overexpression type, the expression levels of all three genes were improved more than 3.6-fold when MYB genes were transiently expressed compared to stable transformation (Figure 6 and Supplementary Figure 5). Consistent with the large accumulation of anthocyanins in transgenic plants, CHS, CHI and F3H expression was significantly activated with increasing gene numbers (Figure 6 and Supplementary Figure 5). Compared to various stresses, normal culture can significantly strengthen the transcriptional levels of CHS, CHI and F3H in transgenic plants (Figure 6 and Supplementary Figure 5).

Regarding the change in the anthocyanin contents of the transgenic plants, the expression of F3′H, F3′5′H, DFR, ANS and UFGT in anthocyanin accumulation was significantly modulated by 4. 7-, 8. 2-, 8. 5-, 11.8- and 3.6-fold, respectively, relative to wild-type plants. But FLS expression was not significantly controlled by anthocyanin content change (Figure 7 and Supplementary Figures 6–8). A purple color provided the highest F3′H, F3′5′H, DFR, ANS and UFGT expression after overexpressing MYB, while the expression of FLS was not affected (Figure 7 and Supplementary Figures 6–8). With the exception of FLS expression, a deeper color of overexpression plants resulted in a significant enhancement in the expression of the late-stage genes (F3′H, F3′5′H, DFR, ANS, UFGT), which was upregulated by at least 4.8-fold compared to the NC plants (Figure 7 and Supplementary Figures 6–8). Correspondingly, the transcriptional levels of F3′5′H, DFR and ANS were significantly decreased by the light color of TC plants (Figure 7 and Supplementary Figure 7). We found that the expression of anthocyanin-specific biosynthesis genes (F3′H, F3′5′H, DFR, ANS, and UFGT) was associated with the number of visual tissues of overexpression plants, and multiple tissues resulted in the transcriptional level of F3′5′H, ANS, and UFGT having a greater effect than one (Figure 7 and Supplementary Figures 6B, 7, 8). F3′H, F3′5′H, DFR, and UFGT showed the highest expression levels across roots in TC plants, while FLS and ANS exhibited higher expression in petal tissues and leaves, respectively (Figure 7 and Supplementary Figures 6–8).

Regarding donor plants, FLS, F3′5′H, ANS and UFGT were significantly upregulated by MYB genes from monocot plants compared to dicots, but F3′H and DFR showed the opposite expression pattern (Figure 7 and Supplementary Figures 6–8). Overexpressing MYB genes from Solanum caused the upregulation of F3′H, DFR, ANS and UFGT expression by 39. 1-, 107. 0-, 50. 3-, and 16.2-fold, respectively (Figure 7 and Supplementary Figures 6B, 8). The transcription of F3′H, DFR and ANS was induced in monocot receiver plants (Figure 7 and Supplementary Figure 6B). In the transgenic MYB-overexpressing Solanum, the expression of F3′5′H, DFR and UFGT relative to NC plants was the most upregulated by 39. 6-, 16. 9-, and 10.1-fold, respectively (Figure 7A and Supplementary Figures 7, 8). The effects of MYB overexpression on FLS, F3′H, F3′5′H, DFR, ANS and UFGT expression depends on the expression system (Figure 7 and Supplementary Figures 6–8). FLS, F3′H and UFGT were significantly upregulated in homologous systems compared with heterologous systems, but the opposite expression pattern was shown by F3′5′H, DFR and ANS (Figure 7 and Supplementary Figures 6–8). Additionally, transient transfection of plants seemed to directly correlate with anthocyanin biosynthetic structural genes (FLS, F3′H, F3′5′H, DFR, ANS, UFGT), wherein F3′5′H and DFR expression was more than 4.3- and 2.9-fold higher than that of stably transformed plants (Figure 7 and Supplementary Figures 6–8). Consistent with the large accumulation of anthocyanins in transgenic plants, multiple gene transfers induced more F3′H, F3′5′H, DFR, ANS, and UFGT expression (Figure 7 and Supplementary Figures 6B, 7, 8). Under culture conditions, normal culture significantly increased the transcriptional levels of FLS, F3′H, F3′5′H, DFR, ANS, and UFGT in transgenic plants (Figure 7 and Supplementary Figures 6–8).

The relative expression levels of LAR and ANR were significantly upregulated, causing increased anthocyanin content in transgenic plants as compared to other anthocyanin content changes (Figure 8). Deep colors, such as red, induced higher expression of LAR and ANR than light colors (Figure 8). One organ color in transgenic plants provided higher expression of LAR and ANR than multiple tissues, which is different from anthocyanins (Figure 8). Regarding the sampled organs, ANR and LAR showed the highest expression levels across other tissues in transgenic plants overexpressing MYB (Figure 8). For donor plants, LAR and ANR were notably upregulated in MYB genes from dicot plants compared with monocot plants (Figure 8). Interestingly, overexpressing MYB genes from Solanum showed the highest impacts on LAR, and significant impacts of ANR expression were observed for Vitis (Figure 8). Recipient plants and the type of expression systems were also important. Higher LAR and ANR expression were observed in the transgenic MYB-overexpressing Nicotiana, but a large difference in those genes was demonstrated by heterologous or homologous expression (Figure 8). The effect of transformation on the expression of LAR and ANR was not consistent across overexpression type and number of gene transfers. The LAR expression was high in transient transfection and single gene transformation and the ANR expression was high in stable overexpression and two genes of TC plants (Figure 8). Growth conditions were also found to alter the expression levels of LAR and ANR, which were significantly upregulated in transgenic plants grown in a normal environment (Figure 8).